L-arabinose fermenting yeast

ABSTRACT

An L-arabinose utilizing yeast strain is provided for the production of ethanol by introducing and expressing bacterial araA, araB and araD genes, L-arabinose transporters are also introduced into the yeast to enhance the uptake of arabinose. The yeast carries additional genomic mutations enabling it to consume L-arabinose, even as the only carbon source, and to produce ethanol. A yeast strain engineered to metabolize arabinose through a novel pathway is also disclosed. Methods of producing ethanol include utilizing these modified yeast strains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/912,493, filed Oct. 24, 2007, which is a national stage entry ofInternational Application No. PCT/US07/64330, filed Mar. 19, 2007, whichclaims priority to U.S. Provisional Application No. 60/810,562, filedJun. 1, 2006. The contents of each application listed above areincorporated by reference in their entirety.

CONTRACTUAL ORIGIN

The United States Government has rights in this invention under ContractNo. DB-AC36-99GO010337 between the United States Department of Energyand the National Renewable Energy Laboratory, a Division of the MidwestResearch Institute.

BACKGROUND

Fuel ethanol is a suitable alternative to fossil fuels. Ethanol may beproduced from plant biomass, which is an economical and renewableresource that is available in large amounts. Examples of biomass includeagricultural feedstocks, paper wastes, wood chips and so on. The sourcesof biomass vary from region to region based on the abundance of naturalor agricultural biomass that is available in a particular region. Forexample, while sugar cane is the primary source of biomass used toproduce ethanol in Brazil, corn-derived biomass, corn starch is a largesource of biomass to produce ethanol in the United States. Otheragricultural feedstocks include, by way of example: straw; grasses suchas switchgrass; grains; and any other lignocellulosic or starch-bearingmaterial.

A typical biomass substrate contains from 35-45% cellulose, 25-40%hemicellulose, and 15-30% lignin, although sources may be found thatdeviate from these general ranges. As is known in the art, cellulose ispolymer of glucose subunits, and hemicellulose contains mostly xylose.Arabinose is also a significant fermentable substrate that is found inbiomass, such as corn fiber and many herbaceous crops in varyingamounts. Other researchers have investigated the utilization ofarabinose and hemicellulose, as reported by Hespell, R. B. 1998.Extraction and characterization of hemicellulose from the corn fiberproduced by corn wet-milling processes. J. Agric Food Chem.46:2615-2619, and McMillan, J. D., and B. L. Boynton. 1994. Arabinoseutilization by xylose-fermenting yeasts and fungi. Appl. Biochem.Biotechnol. 45-46:569-584. The two most abundant types of pentose thatexist naturally are D-xylose and L-arabinose.

It is problematic that most of the currently available ethanol-producingmicroorganisms are only capable of utilizing hexose sugar, such asglucose. This is confirmed by a review of the art, such as is reportedby Barnett, J. A. 1976. The utilization of sugars by yeasts. Adv.Carbohydr. Chem. Biochem. 32:125-234. Many types of yeast, especiallySaccharomyces cerevisiae and related species, are very effective infermenting glucose-based feedstocks into ethanol through anaerobicfermentation. However, these glucose-fermenting yeasts are unable toferment xylose or L-arabinose, and are unable to grow solely on thesepentose sugars. Although other yeast species, such as Pichia stipitisand Candida shehatae, can ferment xylose to ethanol, they are not aseffective as Saccharomyces for fermentation of glucose and have arelatively low level of ethanol tolerance. Thus, the present range ofavailable yeast are not entirely suitable for large scale industrialproduction of ethanol from biomass.

Most bacterial, including E. coli and Bacillus subtilis, utilizeL-arabinose for aerobic growth, but they do not ferment L-arabinose toethanol. These and other microorganisms, such as Zymononas mobilis, havealso been genetically modified to produce ethanol from hexose orpentose. This has been reported, for example, in Deanda, K., M. Zhang,C. Eddy, and S. Picatagglo, 1906, Development of an arabinose-fermentingZymomonas mobilis strain by metabolic pathway engineering. Appl.Environ. Microbiol. 62:4465-4470; and Zhang, M., C. Eddy, K. Deanda, M.Finkelstein, and S. Picataggio, 1995 Metabolic engineering of a pentosemetabolism pathway in ethanologenic Zymomonas mobilis. Science267:240-243. However, it remains the case that the low alcohol toleranceof these non-yeast microorganisms limits their utility in the ethanolindustry.

Much effort has been made over the last decade or so, without trulyovercoming the problem of developing new strains that ferment xylose togenerate ethanol. Such efforts are reported, for example, in Kotter, P.,R. Amore, C. P. Hollenberg, and M. Ciriacy. 1990. Isolation andcharacterization of the Pichia stipitis xylitol dehydrogenase gene,XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiaetransformant. Curr. Genet. 18:493-500; and Wahlbom, C. F., and B.Hahn-Hagardal. 2002 Recent studies have been conducted on yeast strainsthat potentially ferment arabinose. Sedlak, M., and N. W. Ho. 2001.Expression of E. coli araBAD operon encoding enzymes for metabolizingL-arabinose in Saccharomyces cerevisiae, Enzyme Microb. Technol.28:16-24 discloses the expression of an E. coli araBAD operon encodingenzymes for metabolizing L-arabinose in Saccharomyces cerevisiae.Although this strain expresses araA, araB and araD proteins, it isincapable of producing ethanol.

U.S. patent application Ser. No. 10/983,951 by Boles and Beckerdiscloses the creation of a yeast strain that may ferment L-arabinose.However, the overall yield is relatively low, at about 60% oftheoretical value. The rate of arabinose transport into S. cerevisiaemay be a limiting factor for complete utilization of the pentosesubstrate. Boles and Becker attempted to enhance arabinose uptake byoverexpressing the GAL2-encoded galactose permease in S. cerevisiae.However, the rate of arabinose transport using galactose permease wasstill much lower when compared to that exhibited by non-conventionalyeast such as Kluyveromyces marxianus. Another limitation that may havecontributed to the low yield of ethanol in the modified strain of Beckerand Boles is the poor activity of the L-arabinose isomerase encoded bythe bacterial araA gene. Although Becker and Boles used an araA genefrom B. subtilis instead of one from E. coli, the specific activity ofthe enzyme was still low. Other workers in the field have reported thatlow isomerase activity is a bottleneck in L-arabinose utilization byyeast.

There remains a need for new arabinose-fermenting strains that arecapable of producing ethanol at high yield. There is further a need toidentify novel arabinose transporters for introduction intoSaccharomyces cerevisiae to boost the production of ethanol fromarabinose.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods which aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

The presently disclosed instrumentalities overcome some of the problemsoutlined above and advance the art by providing new yeast strains thatare capable of using L-arabinose to produce ethanol at a relatively highyield. Since the yeast galactose permease may facilitate uptake ofarabinose, any Gal⁺ strain possessing endogenous galactose permeaseactivity may be used as described below. Although S. cerevisiae is usedby way of example, the scope of coverage extends to any organismspossessing endogenous pathways to generate ethanol from arabinose and toorganisms into which components of such arabinose metabolic pathways orarabinose transporters may be introduced. The use of S. cerevisiae ispreferred.

In a brief overview of the recombinant technique, the endogenous yeastaldose reductase (AR) gene is disrupted by replacing the AR codingsequence with the yeast LEU2 gene. Because the yeast aldose reductase isthe first enzyme to metabolize arabinose in yeast, an AR³¹ strain isused to reduce diversion of arabinose to unwanted byproducts and toprevent possible inhibition of the isomerase by arabitol. The bacterialaraA, araB, and araD genes are cloned into appropriate yeast expressionvectors. The expression constructs containing all three ara genes areintroduced in the AR⁻ strain and the transformants were capable ofmaking ethanol from L-arabinose.

In another aspect of this disclosure, two novel arabinose transportergenes, termed KmLAT1 and PgLAT2, have been cloned and characterized fromtwo non-conventional yeast species, Kluyveromyces marxianus and Pichiaguilliermondii (also known as Candida guilliermondii), respectively.Both Kluyveromyces marxianus and Pichia guilliermondii are efficientutilizers of L-arabinose, which renders them ideal sources for cloningL-arabinose transporter genes.

The KmLAT1 gene may be isolated using functional complementation of anadapted S. cerevisiae strain that could not grow on L-arabinose becauseit lacked sufficient L-arabinose transport activity. KmLat1 protein hasa predicted length of 556 amino acids encoded by a single ORF of 1668bp. It is a transmembrane protein having high homology to sugartransporters of many different yeast species. When KmLat1 is expressedin S. cerevisiae, transport assays using labeled L-arabinose show thattills transporter has the kinetic characteristics of a low affinityarabinose transporter, with K_(m)=230 mM and V_(max)=55 nmol/mg·min.Transport of L-arabinose by KmLat1 is not significantly inhibited bycommon uncoupling agents but is out-competed by glucose, galactose,xylose, and maltose.

The PgLAT2 gene may be isolated using the technique of differentialdisplay from Pichia guilliermondii. The PgLAT2 gene has an ORF of 1617nucleotides encoding a protein with a predicted length of 539 aminoacids. When PgLAT2 is expressed in S. cerevisiae, transport assays showthat this transporter has almost identical L-arabinose transportkinetics as that of wildtype Pichia guilliermondii. The PgLat2transporter when expressed in S. cerevisiae has a K, of 0.07 mM andV_(max) of 18 nmol/mg-min for L-arabinose transport. Inhibitionexperiments show significant inhibition of the PgLat2 transporter byprotonophores (e.g., NaN₃, DNP, and CCP) and H+-adenosine triphosphatase(ATPase) inhibitors (e.g., DESB and DCCD) similar to inhibition inwildtype P. guilliermondii. Competition experiments show thatL-arabinose uptake by the PgLat2 transporter is inhibited by glucose,galactose, xylose and to a lesser extent by maltose.

The transport kinetics of S. cerevisiae Gal2 have been measured andcompared to those of KmLat1. The S. cerevisiae GAL2 gene (SEQ ID NO 5)under control of a TDH3 promoter exhibits 28 times greater (8.9nmol/mg·mm) L-arabinose transport rate as compared to GAL2 gene undercontrol of a ADH1 promoter. The GAL2-encoded permease (SEQ ID NO 6)shows a K, of 550 mM and a V_(max) of 425 nmol/mg·min for L-arabinosetransport and a K_(m) of 25 mM and a V_(max) of 76 nmol/mg·min forgalactose transport. Although L-arabinose transport by both KmLAT1 andGAL2 encoded permeases is out-competed by glucose or galactose, theinhibitory effects of glucose or galactose are greater on the GAL2encoded permease than on the KmLAT1 encoded transporter.

It is farther disclosed here that a S. cerevisiae strain may betransformed with different combinations of the KmLAT1 and PgLAT2transporter genes and a plasmid carrying the GAL2 gene native to S.cerevisiae. The doubling time for the PgLat2p and Gal2p co-expressingcells grown on L-arabinose is markedly shorter than that of the cellsexpressing only Gal2p, suggesting that L-arabinose uptake may have beenenhanced in these cells. In addition, the PgLat2p and Gal2pco-expressing cells appear to grow to a higher optical density atsaturation, suggesting that this strain may be able to utilize theL-arabinose in the medium more completely. This conclusion is supportedby HPLC analysis which shows significantly less residual L-arabinose inthe culture of cells expressing PgLat2p and Gal2p.

In one embodiment, the transformed strains that carry the newtransporter genes may be further transformed with plasmids carryingthree bacterial genes, araA, araB and araC, which encode proteins thatmay be utilized for arabinose utilization and fermentation. In anotherembodiment, the bacterial genes, araA, araB and araC, may be transformedinto a yeast strain that does not carry any of the new transportergenes.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures of thedrawings. It is intended that the embodiments and figures disclosedherein are to be considered illustrative rather than limiting.

FIG. 1 shows the relationship between KmLAT1 and other transportersbased on the neighbor-joining method (Saitou and Nei 1987).

FIG. 2 shows the DNA (SEQ ID NO. 1) sequence of Kluyveromyces marxianusKmLAT1, and the predicted protein sequence (SEQ ID NO. 2).

FIG. 3 is a schematic presentation of the arabinose metabolic pathway inrecombinant yeast containing proteins encoded by three bacterial genesaraB, araA, and araD.

FIG. 4 shows the library insert from genomic K. marxianus DNAcomplements adapted S. cerevisiae for growth on L-arabinose. Cloninginto the library expression vector is at the indicated BamHI restrictionsites. The black block arrow is the L-arabinose transporter ORFresponsible for complementation (KmLAT1). The block arrow with verticalstripes is the interrupted transporter ORF. The block arrow with thehorizontal stripes is an un-related ORF ligated in place gratuitouslyduring library construction. The Sau3AI restriction site where thetransporter ORF was interrupted is shown. The primer used for PCR basedgenomic walking in K. marxianus is shown.

FIG. 5 shows the growth curve of S. cerevisiae expressing KmLAT1 (Δ),GAL2 (▪) or a control vector (♦) on 2% L-arabinose.

FIG. 6A shows Eadie-Hofstee plot of L-arabinose uptake by KmLat1 (♦) orGal2 (▪) expressed in S. cerevisiae grown on 2% L-arabinose. FIG. 6Bshows comparison of Eadie-Hofstee plots of KmLat1 expressed in S.cerevisiae (♦) and wild type transport activity of K. marxianus (Δ) bothgrown on 2% L-arabinose.

FIG. 7 shows the DNA (SEQ ID NO. 3) sequence of Pichia guilliermondiiPgLAT2, and the predicted protein sequence (SEQ ID NO. 4).

FIG. 8 shows the induction of L-arabinose transport in P.guilliermondii. Uptake of 13 mM labeled sugar was assayed for cellsgrown in minimal media containing 2% L-arabinose, D-galactose orD-xylose. White bars indicated labeled L-arabinose transport. Black barsindicate labeled galactose transport. Bars with vertical stripesindicate labeled xylose transport.

FIG. 9 shows the sugar transport competition analysis in P.guilliermondii grown in minimal L-arabinose medium.

FIG. 10 shows the transport kinetics of L-arabinose by the PgLAT2transporter expressed in S. cerevisiae. Open triangles indicatetransport for wild type P. guilliermondii grown on L-arabinose. Blackdiamonds indicate transport for PgLAT2 expressed in S. cerevisiae grownon L-arabinose.

FIG. 11 comparison of the growth curves in 0.2% L-arabinose for S.cerevisiae cells expressing either Gal2p alone or both Gal2p and PgLAT2.The maximum growth density and growth rate are significantly enhanced inthe strain expressing both Gal2p and PgLAT2.

FIGS. 12A and 12B show the growth curves of BFY001 (parent) (blacksquare) and BFY002 (ΔAR) (black triangle) on glucose and xylulose.

FIGS. 13A, 13B and 13C show the diagrams of the expression plasmids witharaB, araA, and araD genes carrying the His3 selectable marker.

FIGS. 14A, 14B and 14C show the diagrams of the expression plasmids witharaB, araA, and araD genes carrying the Ura3 selectable marker.

FIG. 15 shows histogram with the result of ethanol production inwhole-cell fermentation using yeast cells expressing the three bacterialgenes araB, araA, and araD.

FIGS. 16A, 16B and 16C show histogram with the result of ethanolproduction in cell-free fermentation using yeast cells expressing thethree bacterial genes araB, araA, and araD.

FIGS. 17A, 17B and 17C plot ethanol production using yeast cellsexpressing the three bacterial genes araB, araA, and araD, as a functionof the incubation time in a cell-free fermentation system.

FIG. 18 shows histogram with the result of ethanol production incell-free fermentation using yeast cells expressing the three bacterialgenes araB, araA, and araD.

FIG. 19 plots ethanol production using yeast cells expressing the threebacterial genes araB, araA, and araD, under single- and mixed-sugarfermentations.

DETAILED DESCRIPTION

There will now be shown and described methods for producing transgenicyeast that we capable of metabolizing arabinose and producing ethanol.In the discussion below, parenthetical mention is made to publicationsfrom the references section for a discussion of related procedures thatmay be found useful from a perspective of one skilled in the art. Thisis done to demonstrate what is disclosed by way of nonlimiting example.

The following definitions arc provided to facilitate, understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure:

“Amino acid” refers to any of the twenty naturally occurring amino acidsas well as any modified amino acid sequences. Modifications may includenatural processes such as postranslational processing, or may includechemical modifications which are known in the art. Modifications includebut are not limited to: phosphorylation, ubiquitination, acetylation,amidation, glycosylation, covalent attachment of flavin,ADP-ribosylation, cross linking, iodination, methylation, and the like.

“Antibody” refers to a generally Y-shaped molecule having a pair ofantigen binding sites, a hinge region and a constant region. Fragmentsof antibodies, for example an antigen binding fragment (Fab), chimericantibodies, antibodies haying a human constant region coupled to amurine antigen binding region, and fragments thereof, as well as otherwell known recombinant antibodies are included in this definition.

“Antisense” refers to polynucleotide sequences that are complementary totarget “sense” polynucleotide sequence.

“Biomass” refers collectively to organic non-fossil material. “Biomass”in the present disclosure refers particularly to plant material that isused to generate fuel, such as ethanol. Examples of biomass includes butare not limited, to corn fiber, dried distiller's grain, jatropha,manure, meal and bone meal, miscanthus, peat, plate waste, landscapingwaste, maize, rich hulls, silage, stover, maiden grass, switchgrass,whey, and bagasse from sugarcane.

“Complementary” or “complementarity” refers to the ability of apolynucleotide in a polynucleotide molecule to form a base pair withanother polynucleotide in a second polynucleotide molecule. For example,the sequence A-G-T is complementary to the sequence T-C-A.Complementarity may be partial, in which only some of thepolynucleotides match according to base pairing, or complete, where allthe polynucleotide match according to base pairing.

The term “derivative” refers to compounds that are derived from apredecessor compound by way of chemical or physical modification. Forexample, a compound is a sugar derivatives if it is formed byoxidization of one or more terminal groups to carboxylic acids, byreduction of a carbonyl group, by substitution of hydrogen(s), aminogroup(s), thiol group(s), etc, for one or more hydroxyl groups on asugar, or if it is formed by phosphorylation on a sugar molecule.

“Expression” refers to transcription and translation occurring within ahost cell. The level of expression of a DNA molecule in a host cell maybe determined on the basis of either the amount of corresponding mRNAthat is present within the cell or the amount of DNA molecule encodedprotein produced by the host cell (Sambrook et al., 1989, Molecularcloning: A Laboratory Manual, 18.1-18.88).

“Fusion protein” refers to a first protein attached to a second,heterologous protein. Preferably, the heterologous protein is fused viarecombinant DNA techniques, such that the first and second proteins arcexpressed in frame. The heterologous protein may confer a desiredcharacteristic to the fusion protein, for example, a detection signal,enhanced stability or stabilization of the protein, facilitatedoligomerization of the protein, or facilitated purification of thefusion protein. Examples of heterologous proteins useful as fusionproteins include molecules having full-length or partial protein insequence of KmLat1 or PgLat2. Further examples include peptide tags suchas histidine tag (6-His), leucine zipper, substrate targeting moieties,signal peptides, and the like. Fusion proteins are also meant toencompass variants and derivatives of KmLat1 or PgLat2 polypeptides thatare generated by conventional site-directed mutagenesis and more moderntechniques such as directed evolution, discussed infra.

“Genetically engineered” refers to any recombinant DNA or RNA methodused to create a prokaryotic or eukaryotic host ceil that expresses aprotein at elevated levels, at lowered levels, or in a mutated form. Inother words, the host cell has been transacted, transformed, ortransduced with a recombinant polynucleotide molecule, and thereby beenaltered so as to cause the cell to alter expression of the desiredprotein. Methods and vectors for genetically engineering host cells arewell known in the art; for example various techniques are illustrated inCurrent Protocols In Molecular Biology, Ausubel el al., eds. (Wiley &Sons, New York, 1988, and quarterly updated). Genetic engineeringtechniques include but are not limited to expression vectors, targetedhomologous recombination and gene activation (see, for example, U.S.Pat. No. 5,272,071 to Chappel) and trans activation by engineeredtranscription factors (see, for Example, Segal, et al., 1999, Proc NatlAcad Sci USA 96(6):2758-63). Genetic engineering also encompasses anymutagenesis techniques wherein a cell is exposed to chemicals to induceerrors in DNA replication or to accelerate gene recombination. The term“spontaneous mutation” refers to mutations that occurs at a much lowerrate as a result of genetic recombination or DNA replication errors thatoccur naturally from generation to generation.

“Heterologous” refers to DNA, RNA and/or polypeptides derived fromdifferent organisms or species, for example a bacteria polypeptide isheterologous to yeast.

“Homology” refers to a degree of similarity between polynucleotides,haying significant effect on the efficiency and strength ofhybridization between polynucleotide molecules. The term also refers, toa degree of similarity between polypeptides. Two polypeptides havinggreater than or equal to about 60% similarity are presumptivelyhomologous.

“Host,” “Host cell” or “host cells” refers to cells expressing aheterologous polynucleotide molecule. The term “heterologous” meansnon-native. For instance, when a gene that is not normally expressed inan organism is introduced and expressed in that host organism, such anexpression is heterologous. Host cells of the present disclosure expresspolynucleotides encoding KmLAT1 or PgLAT2 or a fragment thereof.Examples of suitable host cells useful in the present disclosureinclude, but are not limited to, prokaryotic and eukaryotic cells.Specific examples of such cells include bacteria of the generaEscherichia, Bacillus and Salmonella, as well as members of the generaPseudomonas, Streptomyces, and Staphylococcus; fungi, particularlyfilamentous fungi such as Trichoderma and Aspergillus, Phanerochaetechrysosporium and other white not fungi; also other fungi includingFusaria, molds, and yeast including Saccharomyces sp., Pichia sp., andCandida sp. and the like; plants e.g. Arabidopsis, cotton, barley,tobacco, potato, and aquatic plants and the like; SF9 insect cells(Summers and Smith, 1987, Texas Agriculture Experiment Station Bulletin,1555), and the like. Other specific examples include mammalian cellssuch as human embryonic kidney cells (293 cells), Chinese hamster ovary(CHO) cells (Puck et al., 1958, Proc. Natl. Acad. Sci. USA 60,1275-1281), human cervical carcinoma cells (HELA) (ATCC CCL-2), humanliver cells (Hep G2) (ATCC HB8065), human breast cancer cells (MCF-7)(ATCC HTB22), human colon carcinoma cells (DLD-1) (ATCC CCL 221), Daudicells (ATCC CRL-213), murine myeloma cells such as P3/NSI/1-Ag4-1 (ATCCTIB-18), P3X63Ag8 (ATCC TIB-9), SP2/0-Ag14 (ATCC CRL-1581) and the like.The most preferred host is Saccharomyces cerevisiae.

“Hybridization” refers to the pairing of complementary polynucleotidesduring an annealing period. The strength of hybridization between twopolynucleotide molecules is impacted by the homology between the twomolecules, stringency of the conditions involved, the meltingtemperature of the formed hybrid and the G:C ratio within thepolynucleotides.

“Identity” refers to a comparison of two different DNA or proteinsequences by comparing pairs of nucleic acid or amino acids within thetwo sequences. Methods for determining sequence identity are known. See,for example, computer programs commonly employed for this purpose, suchas the Gap program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, Madison Wis.),that uses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math.,2:482-489.

“Isolated” refers to a polynucleotide or polypeptide that has beenseparated from at least one contaminant (polynucleotide or polypeptide)with which it is normally associated. For example, an isolatedpolynucleotide or polypeptide is in a context or in a form that isdifferent from that in which it is found in nature.

“Nucleic acid sequence” refers to the order or sequence ofdeoxyribonuclrotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alonga polypeptide chain. The deoxyribonucleotide sequence thus codes for theamino acid sequence.

“Polynucleotide” refers to a linear sequence of nucleotides. Thenucleotides may be ribonucleotides, or deoxyribonucleotides, or amixture of both. Examples of polynucleotides in this context includesingle and double stranded DNA, single and double stranded RNA, andhybrid molecules having mixtures of single and double stranded DNA andRNA. The polynucleotides may contain one or more modified nucleotides.

“Protein,” “peptide,” and “polypeptide” are used interchangeably todenote an amino acid polymer or a set of two or more interacting orbound amino acid polymers.

“Purify,” or “purified” refers to a target protein makes up for at lastabout 90% of a composition. In other words, it refers to a targetprotein that is free from at least 5-10% of contaminating proteins.Purification of a protein from contaminating proteins may beaccomplished using known techniques, including ammonium sulfate orethanol precipitation, acid precipitation, heat precipitation, anion orcation exchange chromatography, phosphocelluse chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography, size-exclusion chromatography, andlectin chromatography. Various protein purification techniques areillustrated in Current Protocols in Molecular Biology, Ausubel et al.,eds. (Wiley & Sons, New York, 1988, and quarterly updates).

“Selectable marker” refers to a marker that identifies a cell as havingundergone a recombinant DNA or RNA event. Selectable markers include,for example, genes that encode antimetabolite resistance such as theDHFR protein that confers resistance to methotrexate (Wigler et al,1980, Proc Natl Acad Sci USA 77:3567; O'Hare et al., 1981, Proc NatlAcad Sci USA, 78:1527), the GPT protein that confers resistance tomycophenolic acid (Mulligan & Berg, 1981, PNAS USA, 78:2072), theneomycin resistance marker that confers resistance to the aminoglycosideG-418 (Calberre-Garapin et al., 1981, J Mol Biol, 150:1), the Hygroprotein that confers resistance to hygromycin B (Santerre et. al., 1984,Gene 30:147), and the Zeocin™ resistance marker (Invitrogen). Inaddition, the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes may be employed in tk⁻ hgprt⁻ and aprt⁻cells, respectively.

“Transform” means the process of introducing a gene into a host cell.The gene may be foreign in origin, but the gene may also derive from thehost. A transformed host cell is termed a “transformant.” The introducedgene may be integrated onto the chromosome of the host, or the gene mayremain on a stand-alone vector independent of the host chromosomes.

“Variant”, as used herein, means a polynucleotide or polypeptidemolecule that differs from a reference molecule. Variants may includenucleotide changes that result in amino acid substitutions, deletions,fusions, or truncations in the resulting variant polypeptide whencompared to the reference polypeptide.

“Vector,” “extra-chromosomal vector” or “expression vector” refers to afirst polynucleotide molecule, usually double-stranded, which may haveinserted into it a second polynucleotide molecule, for example a foreignor heterologous polynucleotide. The heterologous polynucleotide moleculemay of may not be naturally found in the host cell, and may be, forexample, one or more additional copy of the heterologous polynucleotidenaturally present in the host genome. The vector is adapted fortransporting the foreign polynucleotide molecule into a suitable hostcell. Once in the host cell, the vector may be capable ofintegrating-into the host cell chromosomes. The vector may optionallycontain additional elements for selecting cells containing theintegrated polynucleotide molecule as well as elements to promotetranscription of mRNA from transacted DNA. Examples of vectors useful inthe methods disclosed herein include, but are not limited to, plasmids,bacteriophages, cosmids, retroviruses, and artificial chromosomes.

For purpose of this disclosure, unless otherwise stated, the techniquesused may be found in any of several well-known references, such as:Molecular Cloning: A Laboratory Manual (Sambrook et al. (1989) Molecularcloning: A Laboratory Manual), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991 Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, 3d., (1990) Academic Press, Inc.), PCR Protocols; AGuide to Methods and Applications (Innis et al. (1990) Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) ed. (R. I. Freshney (1987) Liss, Inc., New York,N.Y.), and Gene Transfer and Expression Protocols, pp 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.).

Unless otherwise indicated, the term “yeast,” “yeast strain” or “yeastcell” refers to baker's yeast, Saccharomyces cerevisiae. Other yeastspecies, such as Kluyveromyces marxianus or Pichia guilliermondii, arereferred to as non-conventional yeast in this disclosure. Strains of S.cerevisiae, depository information, and plasmids used for thisdisclosure are listed in Table 1, 2 and Table 3, respectively, The yeastKluyveromyces marxianus CBS-1089 is obtained from the Centraalbureauvoor Schimmelcultures (CBS) collection. Pichia guilliermondii NRRLY-2075 is obtained from the Agricultural Research Service CultureCollection (NRRL).

TABLE 1 S. cerevisiae Strains Used in this Disclosure Strain GenotypePlasmids BFY001 MATa ura3-52 trp1-Δ63 his3-Δ200 leu2-Δ1 BFY002 MATaura3-52 trp1-Δ63 his3-Δ200 leu2-Δ1 yhr104w::LEU2 BFY507 MATa ura3-52trp1-Δ63 his3-Δ200 leu2-Δ1 p138, p42 yhr104w::LEU2 adapted for growth onL-arabinose BFY518 same as BFY507 p138 BFY566 same as BFY518 p138, p171BFY590 same as BFY518 gal2dcHIS3 p138 BFY597 same as BFY590 p138, p42BFY598 same as BFY590 p138, p187 BFY012 same as BFY002 pBFY004, pBFY013,pBFY012 BFY013 same as BFY002 pBFY007, pBFY016, pBFY014 BFY014 same asBFY002 pBFY007, pBFY015, pBFY017 BFY015 same as BFY002 pBFY005, pBFY016,pBFY019 BFY016 same as BFY002 pBFY005, pBFY018, pBFY017 BFY017 same asBFY002 pBFY009, pBFY018, pBFY014 BFY018 same as BFY002 pBFY009, pBFY015,pBFY019 BFY057 MATa his3D1 leu2D0 ura3D0 met15D0 gal80D::G418yhr104w::LEU2 BFY534 same as BFY057 p144, p165 BFY535 same as BFY057p144, pBFY13 BFY605 same as BFY590 p244 BFY625 MATa his3Δ1 leu2Δ0 ura3Δ0trp1Δ pBFY12, pBFY13, p138 met15Δ0 gal80Δ::G418 adapted for growth onL-arabinose BFY626 Same as BFY625 pBFY12, p138, p204

Yeast strains may be grown on liquid or solid media with 2% agar forsolid media. Where appropriate, some amino acids or nucleic acids arepurposely left out from the media for plasmid maintenance. Growthconditions are typically 30° C. unless otherwise indicated, with shakingin liquid cultures. Anaerobic conditions are generally more favorable tometabolize the various sugars to ethanol.

A number of the yeast strains listed in Table 1 have been deposited atthe American Type Culture Collection (ATCC) in accordance with theprovisions of the Budapest Treatey on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedures. Ineach instance, the yeast strain was deposited by the inventors listedherein on Mar. 16, 2007 at American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110 U.S.A.

TABLE 2 Depository Information Yeast Strain/Accession NumberBFY013/PTA-8258 BFY534/PTA-8257 BFY598/PTA-8256 BFY626/PTA-8255

TABLE 3 Plasmids Used in this Disclosure Plasmid Marker and expressedgenes p42 URA3, GAL2 over-expression p138 TRP1, B. subtilis araA, E.coli araB, E. coli araD p144 E. coli araB, D; B. subtilis araA inpBFY012 p165 HIS3, GAL2 over-expression p171 HIS3, 8.8 kb K. marxianusgenomic DNA fragment p187 URA3, KmLAT1 over-expression plasmid p204HIS3, PgLAT2 over-expression plasmid p244 URA3, PgLAT2 over-expressionplasmid pBFY004 control 2μ vector with PGK promoter, GAL10 terminatorand Trp1 marker pBFY005 E. coli araB in pBFY004 pBFY007 E. coli araA inpBFY004 pBFY009 E. coli araD in pBFY004 pBFY012 control 2μ vector withPGK promoter, GAL10 terminator and Ura3 marker pBFY013 control 2μ vectorwith PGK promoter, GAL10 terminator and His3 marker pBFY014 E. coli araBin pBFY012 pBFY015 E. coli araB in pBFY013 pBFY016 E. coli araD inpBFY013 pBFY017 E. coli araD in pBFY012 pBFY018 E. coli araA in pBFY013pBFY019 E. coli araA in pBFY012

Yeast cells may be grown in rich media YPD or minimum mediaconventionally used in the field. YPD medium contains about 1% yeastextract, 2% peptone and 2% dextrose. Yeast minimum media typicallycontains 0.67% of yeast nitrogen base (“YNB”) without amino acidssupplemented with appropriate amino acids or purine or pyrimidine bases.An amount of sugar, typically 2% unless otherwise indicated, may be usedas carbon source, including glucose (dextrose), galactose, maltose orL-arabinose among others. Adaptation for growth on L-arabinose isperformed as described in, for example, Becker and Boles (2003) withmodifications as detailed in Example 3.

Over-expression plasmids are constructed by cloning the gene forover-expression downstream of the S. cerevisiae PGK1 or TDH3 promoter ina 2μ-based vector. Construction of a DNA library is detailed in theExamples. Note that other like S. cerevisiae prompters can also be usedfor overexpression, including ADH2, PDC1, PGI1, etc.

E. coli cells may be grown in LB liquid media or on LB agar platessupplemented with ampicillin at 100 μg/ml as needed. Transformation ofE. coli DH5α is by electrotransformation according to a protocol byInvitrogen (Invitrogen 11319-019). After transformation, the baterialcells are plated on LB plates containing 100 μg/ml ampicillin forselection. Transformation of S. cerevisiae was performed using aDMSO-enhanced lithium-acetate procedure as described with the followingmodifications (Hill et al., 1991). Cells are harvested and initiallywashed in water. 600 μl of PBG4000 solution is added and 70 μl DMSO isadded just prior to heat shocking. Cells are heat-shocked for 15 min at42°0 C. and the last wash stop is skipped. Cells are resuspended in 10mM TE solution and plated.

Yeast DNA is isolated using the Easy DNA kit according to manufacturer'sprotocol (Invitrogen, K1800-01). DNA manipulations and libraryconstruction are performed as described in Molecular Cloning: ALaboratory Manual (1989), except otherwise specifically indicated inthis disclosure. Plasmids are cured from yeast by growing the strain inrich non-selective media overnight followed by plating on non-selectivemedia. Isolated colonies are replica plated to screen for loss ofselective markers. Plasmid rescue is performed by transforming isolatedyeast DNA into E. coli followed by isolation and characterization, E.coli plasmid isolation is accomplished using plasmid spin mini-prep kitaccording to the manufacturer's manual (Siegen, 27106). PCR-basedchromosomal walking is performed using the Universal GenomeWalker Kit asdescribed (BD Biosciences, K1807-1).

For the transport assays, cells may be grown in minimal mediasupplemented with 20 g/L of L-arabinose. Cells are collected inmid-growth and washed twice before suspension in water at 30 mg/ml.Uptake of L-(1-¹⁴C) arabinose (54 mCi/mmol, Moravek Biochemicals Inc.)or D-(1-¹⁴C) galactose (57 mCi/mmol, Amersham Biosciences) is measuredas previously described by Stambuk et al. (2003). Assays are performedin 30 seconds to maintain initial rates after appropriate experiments toensure uptake is linear for at least 1 minute. Transport activity isdescribed as nano-moles of labeled sugar transported per mg cell dryweight per minute. Inhibition and competition assays are performed aspreviously described by Stambuk et al. (2003).

Sequencing results showed that the KmLAT1 gene contains on ORF of 1668bp in length. The predicted amino acid sequence of KmLAT1 shareshomology With high-affinity glucose transporters, in particular, withHGT1 with high-affinity glucose transporters from non-conventional yeastthan With transporter proteins encoded by the bacterial araE gene orhexose transporters from S. cerevisiae (FIG. 1).

TABLE 4 Properties and similarities of KmLat1 to other sugartransporters. Predicted protein Predicted Degree of (no. of aa/ pl oftransmembrane identity (%)/ Putative function gene no. of kDa) proteinregions similarity (%) Organism of gene product KmLat1 556/61.3 8.22 12— K. marxianus ¹ L-arabinose transporter KlHgt1 551/60.8 5.76 12 77/89K. lactis ² high affinity glucose transporter AEL042Cp 547/59.8 8.82 1265/82 A. gossypii ³ putative hexose transporter DEHA0E01738g 545/61.15.55 12 52/70 D. hansenii ⁴ hexose transporter CaHgt1 545/60.7 8.0512-13 50/71 C. albicans ⁵ putative hexose transporter CaHgt2 545/60.48.48 12-14 51/71 C. albicans ⁶ putative hexose transporter Accessionnumbers: ¹Not yet assigned, ²1346290, ³AEL042C, ⁴DEHA0E01738g,⁵CAA76406, ⁶orf19.3668

Transmembrane regions predicted for KmLat1 and PgLat2 by the softwareTmpred shows 12 transmembrane regions with a larger intercellular loopbetween regions 6 and 7 (FIG. 2) (See Hofmann et al, 1993), typical ofGAL2 and other yeast sugar transporters having 10-12 transmembraneregions (See e.g., Alves-Araujo et al., 2004; Day et al., 2002;Kruckeberg et al., 1996; Pina et al., 2004; and Welerstall et al. 1999).

Like other members of the transporter family, and in particular sugartransporters, KmLat1 and PgLat2 polypeptides are useful in facilitatingthe uptake of various sugar molecules into the cells. It is envisionedthat KmLat1 or PgLat2 polypeptides could be used for other purposes, forexample, in analytical instruments or other processes where uptake ofsugar is required. KmLat1 or PgLat2 polypeptides may be used alone or incombination with one or more other transporters to facilitate themovement of molecules across a membrane structure, which function may bemodified by one skilled in the relevant art, all of which are within thescope of the present disclosure.

The KmLAT1 polypeptides include isolated polypeptides having an aminoacid sequence as shown below in Example 2; and in SEQ ID NO:2, as wellas variants and derivatives, including fragments, having substantialsequence similarity of the amino acid sequence of SEQ ID NO:2 and thatretain any of the functional activities of KmLAT1. PgLAT2 polypeptidesinclude isolated polypeptides having an amino acid sequence as shpwnbelow in Example 5; and in SEQ ID NO:4, as well as variants andderivatives, including fragments, having substantial sequence similarityto the amino acid sequence of SEQ ID NO:4 and that retain any of thefunctional activities of PgLAT2. The functional activities of the KmLAT1or PgLAT2 polypeptides include but are not limited to transport ofL-arabinose across cell membrane. Such activities may be determined, forexample, by subjecting the variant, derivative, or fragment to aarabinose transport assay as detailed, for example in Example 4.

Variants and derivatives of KmLAT1 or PgLAT2 include, for example,KmLAT1 or PgLAT2 polypeptides modified by convalent or aggregativeconjugation with other chemical moieties, such as glycosyl groups,polyethylene glycol (PEG) groups, lipids, phosphate, acetyl groups, andthe like.

The amino acid sequence of KmLAT1 or PgLAT2 polypeptides is preferablyat least about 60% identical, more preferably at least about 70%identical, more preferably still at least about 80% identical, and insome embodiments at least about 90%, 95%, 96%, 97%, 98%, and 99%identical, to the KmLAT1 and PgLAT2 amino acid sequences of SEQ ID NO: 2and SEQ ID NO: 4, respectively. The percentage sequence identity, alsotermed homology (see definition above) may be readily determined, forexample, by comparing the two polypeptide sequences using any of thecomputer programs commonly employed for this purpose; such as the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), whichuses the algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482-489.

Variants and derivatives of the KmLAT1 or PgLAT2 polypeptides mayfurther include, for example, fusion proteins formed of a KmLAT1 orPgLAT2 polypeptide and another polypeptide. Fusion protein may be formedbetween a fragment of the KmLAT1 polypeptide and another polypeptide,such that the fusion protein may retain none or only part of theactivities normally performed by the full-length KmLAT1 or PgLAT2polypeptide. Preferred polypeptides, for constructing the fusion proteininclude those that facilitate purification or oligomerization, or thosethat enhance KmLAT1 or PgLAT2 stability and/or transport capacity ortransport rate for sugars, especially for arabinose. Preferredpolypeptides may also include those that gain enhanced transportcapability when fused with KmLAT1, PgLAT2 or fragments thereof.

KmLAT1 or PgLAT2 variants and derivatives may contain conservativelysubstituted amino acids, meaning that one or more amino acid may bereplaced by an amino acid that does not alter the secondary and/ortertiary structure of the polypeptide. Such substitutions may includethe replacement of an amino acid, by a residue having similarphysicochemical properties, such as substituting one aliphatic residue(Ile, Val, Leu, or Ala) for another, or substitutions between basicresidues Lys and Arg, acidic residues Glu and Asp, amide residues Glnand Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe andTyr. Phenotypically silent amino acid exchanges are described more fullyin Bowie et al., 1990. In addition, functional KmLAT1 or PgLAT2polypeptide variants include those having amino acid substitutions,deletions, or additions to the amino acid sequence outside functionalregions of the protein. Techniques for making these substitutions anddeletions are well known in the art and include, for example,site-directed mutagenesis.

The KmLAT1 or PgLAT2 polypeptides may be provided in an isolated form,or in a substantially purified form. The polypeptides may be recoveredand purified from recombinant cell cultures by known methods, including,for example, ammonium sulfate or ethanol precipitation, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Preferably, proteinchromatography is employed for purification.

A preferred form of KmLAT1 or PgLAT2 polypeptides is that of recombinantpolypeptides expressed by suitable hosts. In one preferred embodiment,when heterologous expression of KmLAT1 or PgLAT2 is desired, the codingsequences of KmLAT1 or PgLAT2 may be modified in accordance with thecodon usage of the host. Such modification may result in increaseprotein expression of a foreign in the host. Furthermore, the hosts maysimultaneously produce other transporters such that multipletransporters are expressed in the same cell, wherein the differenttransporters may form oligomers to transport the same sugar.Alternatively, the different transporters may function independently totransport different sugars. Such recombinant cells may ne useful incrude fermentation processing or in other industrial processing.

KmLAT1 or PgLAT2 polypeptides may be fused to heterologous polypeptidesto facilitate purification. Many available heterologous peptides(peptide tags) allow selective binding of the fusion protein to ablinding partner. Non-limiting examples of peptide tags include 6-His,thioredoxin, hemaglutinin, GST, and the OmpA signal sequence tag. Abinding partner that recognizes and binds to the heterologous peptidemay be any molecule or compound, including metal ions (for example,metal affinity columns), antibodies, antibody fragments, or any proteinor peptide that preferentially binds the heterologous peptide to permitpurification of the fusion protein.

KmLAT1 or PgLAT2 polypeptides may be modified to facilitate formation ofKmLAT1 or PgLAT2 oligomers. For example, KmLAT1 polypeptides may befused to peptide moieties that promote oligomerization, such as leucinezippers and certain antibody fragment polypeptides, for example, Fcpolypeptides. Techniques for preparing these fusion proteins are known,and are described, for example, in WO 99/31241 and in Cosman et. al.,2001. Fusion to an Fc polypeptide offers the additional advantage offacilitating purification by affinity chromatography over Protein A orProtein G columns. Fusion to a leucine-zipper (LZ), for example, arepetitive heptad repeat, often with four or five leucine residuesinterspersed with other amino acids, is described in Landschultz et al.,1988.

It is also envisioned that an expanded set of variants and derivativesof KmLAT1 or PgLAT2 polynucleotides and/or polypeptides may be generatedto select for useful molecules, where such expansion is achieved notonly by conventional methods such as site-directed mutagenesis but alsoby more modern techniques, either independently or in combination.

Site-directed-mutagenesis is considered an informational approach toprotein engineering and may rely on high-resolution crystallographicstructures of target proteins for specific amino acid changes (van denBurg et al. 1998). For example, modification of the amino acid sequenceof KmLAT1 or PgLAT2 polypeptides may be accomplished as is known in theart, such as by introducing mutations at particular locations byoligonucleotide-directed mutagenesis Site-directed-mutagenesis may alsotake advantage of the recent advent of computational methods foridentifying site-specific changes for a variety of protein engineeringobjectives (Hellinga, 1998).

The more modern techniques include, but are not limited to,non-informational mutagenesis techniques (referred to generically as“directed evolution”). Directed evolution, in conjunction withhigh-throughput screening, allows testing of statistically meaningfulvariations in protein conformation (Arnold, 1998). Directed evolutiontechnology may include diversification methods similar to that describedby Crameri et al. (1998), site-saturation mutagenesis, staggeredextension process (StEP) (Zhao et al., 1998), and DNAsynthesis/reassembly (U.S. Pat. No. 5,965,408).

Fragments of the KmLAT1 or PgLAT2 polypeptide maybe used, for example,to generate specific anti-KmLAT1 antibodies. Using known selectiontechniques, specific epitopes may be selected and used to generatemonoclonal or polyclonal antibodies. Such antibodies have utility in theassay of KmLAT1 or PgLAT2 activity as well as in purifying recombinantKmLAT1 or PgLAT2 polypeptides from genetically engineered host cells.

The disclosure also provides polynucleotide molecules encoding theKmLAT1 or PgLAT2 polypeptides discussed above. KmLAT1 or PgLAT2polynucleotide molecules include polynucleotide molecules having thenucleic acid sequence shown in SEQ ID NO:1 and SEQ ID NO:3,respectively; polynucleotide molecules that hybridize to the nucleicacid sequence of SEQ ID NO:1 and SEQ ID NO:3, respectively, under highstringency hybridization conditions (for example, 42°, 2.5 hr., 6×SCC,0.1% SDS); and polynucleotide molecules having substantial nucleic acidsequence identity with the nucleic acid sequence of SEQ ID NO:1 and SEQID NO:3, respectively. It will be appreciated that such polynucleotidemolecules also broadly encompass equivalent substitutions of codons thatmay be translated to produce the same amino acid sequences, truncatedfragments of the polynucelotide molecues, and polynucleotide moleculeswith a high incidence of homology, such as 90%, 95%, 96%, 97%, 98%, or99% or more homology with respect to what is disclosed.

The KmLAT1 or PgLAT2 polynucleotide molecules of the disclosure arepreferably isolated molecules encoding the KmLAT1 or PgLAT2 polypeptidehaving an amino acid sequence as shown in SEQ ID NO:2 and SEQ ID NO:4,respectively, as well as derivatives, variants, and useful fragments ofthe KmLAT1 or PgLAT2 polynucleotide. The KmLAT1 or PgLAT2 polynucleotidesequence may include deletions, substitutions, or additions to thenucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:3, respectively.

The KmLAT1 or PgLAT2 polynucleotide molecule may be cDNA, chemicallysynthesized DNA, DNA amplified by PCR, RNA, or combinations thereof. Dueto the degeneracy of the genetic code, two DNA sequences may differ andyet encode identical amino acid sequences. The present disclosure thusprovides an isolated polynucleotide molecule having a KmLAT1 or PgLAT2nucleic acid sequence encoding KmLAT1 or PgLAT2 polypeptide, wherein thenucleic acid sequence encodes a polypeptide haying the complete aminoacid sequences as shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, orvariants, derivatives, and fragments thereof.

The KmLAT1 or PgLAT2 polynucleotides of the disclosure have a nucleicacid sequence that is at least about 60% identical to the nucleic acidsequence shown in SEQ ID NO:1 and SEQ ID NO:3, respectively, in someembodiments at least about 70% identical to the nucleic acid sequenceshown in SEQ ID NO:1 and SEQ ID NO:3, respectively, at least, about 80%identical to the nucleic acid sequence shown in SEQ ID NO:1 and SEQ IDNO:3, respectively, and in other embodiments al least about 90%-95%,96%, 97%, 98%, 99%% identical to the nucleic acid sequence shown in SEQID NO:1 and SEQ ID NO:3, respectively. Nucleic acid sequence identity isdetermined by known methods, for example by aligning two sequences in asoftware program such as the BLAST program (Altschul, S. F et al. (1990)J. Mol. Biol. 215:403-410, from the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov//BLAST/).

The KmLAT1 or PgLAT2 polynucleotide molecules of the disclosure alsoinclude isolated polynucleotide molecules having a nucleic acid sequencethat hybridizes under high stringency conditions (as defined above) to athe nuclei acid sequence shown in SEQ ID NO:1 and SEQ ID NO:3,respectively. Hybridization of the polynucleotide is to at least about15 contiguous nucleotides, oral least about 20 contiguous nucleotides,and in other embodiments at least about 30 contiguous nucleotides, andin still other embodiments at least about 100 contiguous nucleotides ofthe nucleic acid sequence shown in SEQ ID NO:1 and SEQ ID NO:3,respectively.

Useful fragments of the KmLAT1 or PgLAT2 polynucleotide moleculesdescribed herein, include probes and primers. Such probes and primersmay be used, for example, in PCR methods to amplify and detect thepresence of KmLAT1 or PgLAT2 polynucleotides in vitro, as well as inSouthern and Northern blots for analysis of KmLAT1 or PgLAT2. Cellsexpressing the KmLAT1 or PgLAT2 polynucleotide molecules may also beidentified by the use of such probes. Methods for the production and useof such primers and probes are known. For PCR, 5′ and 3′ primerscorresponding to a region at the termini of the KmLAT1 or PgLAT2polynucleotide molecule may be employed to isolate and amplify theKmLAT1 PgLAT2 polynucleotide using conventional techniques,

Other useful fragments of the KmLAT1 or PgLAT2 polynucleotides includeantisense or sense oligonucleotides comprising a single-stranded nucleicacid sequence capable of binding to a target KmLAT1 or PgLAT2 mRNA(using a sense strand), or DNA (using an antisense strand) sequence.

The present disclosure also provides vectors containing thepolynucleotide molecules, as well as host cells transformed with suchvectors. Any of the polynucleotide molecules of the disclosure may becontained in a vector, which generally includes a selectable marker andan origin of replication, for propagation in a host. The vectors mayfurther include suitable transcriptional or translational regulatorysequences, such as those derived from a mammalian, fungal, bacterial,viral, or insect genes, operably linked to the KmLAT1 or PgLAT2polynucleotide molecule. Examples of such regulatory sequences includetranscriptional promoters, operators, or enhancers, mRNA ribosomalbinding sites, and appropriate sequences which control transcription andtranslation. Nucleotide sequences are operably linked when theregulatory sequence functionally relates to the DNA encoding the targetprotein. Thus, a promoter nucleotide sequence is operably linked to aKmLAT1 or PgLAT2 DNA sequence if the promoter nucleotide sequencedirects the transcription of the KmLAT1 or PgLAT2 sequence.

Selection of suitable vectors for the cloning of KmLAT1 or PgLAT2polynucleotide molecules encoding the KmLAT1 or PgLAT2 polypeptides ofthis disclosure depends upon the host cell in which the vector will betransformed, and, where applicable, the host cell from which the targetpolypeptide is to be expressed. Suitable host cells for expression ofKmLAT1 or PgLAT2 polypeptides include prokaryotes, yeast, and highereukaryotic cells, each of which is discussed below. Selection ofsuitable combinations of vectors and host organisms is a routine matterfrom a perspective of skill.

The KmLAT1 or PgLAT2 polypeptides to be expressed in such host cells mayalso be fusion proteins that include sequences from other proteins. Asdiscussed above, such regions may be included to allow, for example,enhanced functionality, improved stability, or facilitated purificationof the KmLAT1 or PgLAT2 polypeptide. For example, a nucleic acidsequence encoding a peptide that binds strongly to arabinose may befused in-frame to the transmembrane sequence of the KmLAT1 or PgLAT2polypeptides so that the resulting fusion protein binds arabinose andtransports the sugar across the cell membrane at a higher rate than theKmLAT1 or PgLAT2 transporter.

Suitable host cells for expression of target polypeptides includeprokaryotes, yeast, and higher eukaryotic ceils. Suitable prokaryotichosts to be used for the expression of these polypeptides includebacteria of the genera Escherichia, Bacillus, and Salmonella, as well asmembers of the genera Pseudomonas, Streptomyces, and Staphylococcus.

Expression vectors for use in prokaryotic hosts generally comprise oneor more phenotypic selectable marker genes. Such genes encode, forexample, a protein that confers antibiotic resistance or that suppliesauxotrophic requirement. A wide variety of such vectors are readilyavailable from commercial sources. Examples include pSPORT vectors, pGEMvectors (Promega, Madison, Wis.), pPROEX vectors (LTI, Bethesda, Md.),Bluescript vectors (Stratagene), and pQE vectors (Qiagen).

KmLAT1 or PgLAT2 may also be expressed in yeast host cells from generaincluding Saccharomyces, Pichia, and Kluveromyces. Preferred yeast hostis S. cerevisiae. Yeast vectors will often contain an origin ofreplication sequence from a 2μ yeast plasmid for high copy vectors and aCEN sequence for a low copy number vector. Other sequences on a yeastvector may include an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Vectorsreplicable in both yeast and E. coli (termed shuttle vectors) arepreferred. In addition to the above-mentioned features of yeast vectors,a shuttle vector will also include sequences for replication andselection in E. coli.

Insect host cell culture systems may also be used for the expression ofKmLAT1 or PgLAT2 polypeptides. The target polypeptides are preferablyexpressed using a baculovirus expression system, as described, forexample, in the review by Luckow and Summers, 1988.

The choice of a suitable expression vector for expression of KmLAT1 orPgLAT2 polypeptides will depend upon the host cell to be used. Examplesof suitable expression vectors for E. coli include pET, pUC, and similarvectors as is known in the art. Preferred vectors for expression of theKmLAT1 or PgLAT2 polypeptides include the shuttle plasmid plJ702 forStreptomyces lividans, pGAPZalpha-A, B, G and pPICZalpha-A, B, C(Invitrogen) for Pichia pastoris, and pFE-1 and pFE-2 for filamentousfungi and similar vectors as is known in the art. The vectors preferredfor expression in S. cerevisiae are listed in Table 2.

Modification of a KmLAT1 or PgLAT2 polynucleotide molecule to facilitateinsertion into a particular vector (foe example, by modifyingrestriction sites), ease of use in a particular expression system orhost (for example, using preferred host codons), and the like, are knownand are contemplated for use. Genetic engineering methods for theproduction of KmLAT1 or PgLAT2 polypeptides include the expression ofthe polynucleotide molecules in cell free expression systems, in hostcells, in tissues, and in animal models, according to known methods.

This disclosure also provides reagents, compositions, and methods thatare useful for analysis of KmLAT1 or PgLAT2 activity and for assessingthe amount and rate of arabinose transport.

The KmLAT1 or PgLAT2 polypeptides of the present disclosure, in whole orin part, may be used to raise polyclonal and monoclonal antibodies thatare useful in purifying KmLAT1 or PgLAT2, or detecting KmLAT1 or PgLAT2polypeptide expression, as well as a reagent tool for characterizing themolecular actions of the KmLAT1 or PgLAT2 polypeptide. Preferably, apeptide containing a unique epitope of the KmLAT1 or PgLAT2 polypeptideis used in preparation of antibodies, using conventional techniques.Methods for the selection of peptide epitopes and production ofantibodies are known. See, for example, Antibodies: A Laboratory Manual,Harlow and Land (eds.), 1988 Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Monoclonal Antibodies, Hybridomas: A New Dimensionin Biological Analyses, Kennet et al. (eds.), 1980 Plenum Press, NewYork.

Agents that modify, for example, to increase or decrease, KmLAT1 orPgLAT2 transport of arabinose or other sugars may be identified by thetransport assay described in Example 4, for example. Performing thetransport assay in the presence or absence of a test agent permitsscreening of such agents.

The KmLAT1 or PgLAT2 transport activity is determined in the presence orabsence of a test agent and then compared. For instance, a lower KmLAT1transport activity in the presence of the test agent, than in theabsence of the test agent, indicates that the test agent has decreasedthe activity of the KmLAT1. Stipulators and inhibitors of KmLAT1 orPgLAT2 may be used to augment, inhibit, or modify KmLAT1 or PgLAT2transport activity, and therefore may have potential industrial uses asveil as potential use in further elucidation of the molecular actions ofKmLAT1 or PgLAT2.

The KmLAT1 or PgLAT2 polypeptide of the disclosure is an effectivearabinose transporter. In the methods of the disclosure, the sugartransporting effects of KmLAT1 or PgLAT2 are achieved by mixing cellsexpressing KmLAT1 or PgLAT2 with pure sugar or sugar-containing biomass.KmLAT1 or PgLAT2 may also be used in a cell-free system. KmLAT1 orPgLAT2 may be used under other conditions, for example, at elevatedtemperatures or under acidic pH. Other methods of using KmLAT1 or PgLAT2to transport sugar, especially arabinose, for fermentation, areenvisioned to be within the scope of what is disclosed, KmLAT1 or PgLAT2polypeptides may be used in any known application currently utilizing asugar transporter, all of which are within the scope of this disclosure.

It is shown in this disclosure that Gal2p is an effective L-arabinosetransporter at high concentrations of arabinose, whereas KmLAT1 orPgLAT2 may be more effective at different concentrations of L-arabinose.Combination of the Gal2p and the two new transporters fromnon-conventional yeast may be employed to provide complementarytransport into S. ceretfsiae of L-arabinose down to very low residualconcentration of arabinose.

It is shown here that combinatorial expression of Gal2p, KmLAT1 andPgLAT2 may enhance the overall rate and extent of arabinose utilizationby recombinant S. cereviciae cells expressing these transporters. Asshown in Example 8, the doubling time for S. cereviciae strainexpressing both PgLAT2 and Gal2p is shorter than S. cereviciae cellsexpressing Gal2p alone (15 hours vs. 19 hours), suggesting thatL-arabinose uptake may be enhanced by the synergistic effect of PgLAT2and Gal2p in these cells. Moreover the PgLAT2 expressing strain appearsto grow to a higher overall optical density at saturation, suggestingthat this strain was able to utilize the carbon source (L-arabinose) inthe medium more completely. This hypothesis is supported by HPLCanalysis of the final culture media (Table 5) which indicates that thereis significantly less residual L-arabinose in the culture of cellsexpressing Gal2p and PgLAT2 than in the culture of those expressingGal2p alone. Thus, heterologous expression of either or both KmLAT1 andPgLAT2 in S. cereviciae may enhance arabinose utilization byfacilitating arabinose transport when the concentration of arabinose isrelatively low.

TABLE 5 Doubling times and HPLC Measurement of Residual ArabinoseConcentration in Cultures Described in FIG. 11. Transporters DoublingL-arabinose Flask Expressed Time (hours) Final OD₆₀₀ (g/L) by HPLC 1Gal2p only 19.2 0.72 0.68 2 18.6 0.72 0.67 3 Gal2p + PgLat2 15.0 0.850.49 4 14.8 0.85 0.48 *starting L-arabinose concentration 1.89 g/L andmedia without L-arabinose had an undetectable level (<0.1 g/L). ND = notdetermined.

L-arabinose metabolism in bacteria involves three enzymes: L-arabinoseisomerase (araA), L-ribulokinase (araB), and L-ribulose-5-p 4-epimerase(araD), which may be collectively referred to as the “araBAD” proteinsin this disclosure. The genes encoding these three enzymes may bereferred to as the “araBAD” genes in this disclosure. The combinedaction of these three bacterial proteins, convert L-arabinose toXylulose-5-phosphate (See FIG. 3). S. cerevisiae contains the pathway toutilize and ferment the final product xylulose-5-phosphate and produceethanol under certain conditions (see FIG. 3).

S. cerevisiae strain to be used to construct an arabinose fermentingyeast strain preferably possesses Gal⁺ phenotype. A Gal⁺ strain islikely to express galactose permease which may facilitate the uptake ofarabinose by S. cerevisiae.

S. cerevisiae typically possesses endogenous aldose reductase (“AR”)activity, which may divert arabinose to a pathway different from the onethat may lead to the production of ethnaol through the action of thebacterial araBAD proteins. Moreover, the arabitol generated by the ARprotein may inhibit the isomerase encoded by araA. In order to increasethe overall yield of ethanol from arabinose, it is preferable to use anAR-deficient strain to construct the arabinose fermenting yeast of thepresent disclosure. The AR-deficient strain may be obtained by screeningfor spontaneous mutations, or preferably by targeted gene disruption ormutation. An example of such gene disruption is detailed in Example 10.

As shown in FIG. 3, the engineered pathway utilizing bacterial araBADconverts L-arabinose to xylulose-5-P that S. cerevisiae can convert toethanol using endogenous enzymatic activities. It is thus desirable toensure that the arabinose metabolic pathway starting from xylulose toethanol remains intact in the AR-deficient strain. This may be tested bycomparing the growth of the AR-deficient strain with its parental strainon glucose or xylulose. If both strains proliferate at about the samerate on glucose or xylulose, it is likely that the AR gene disruptionevent has not negatively impacted the catabolism of glucose of xylulose.

The present disclosure also provides a new method to measure arabinoseuptake by yeast cells. Traditionally, L-arabinose transport is measuredby using radio-labeled substrate. Since aldose reductase, which convertsL-arabinose to arabitol, is cytosolic, it is possible to use theformation of new arabitol as an indicator of arabinose uptake. Higherlevels of arabitol indicates higher uptake of L-arabinose. To confirmthe validity of this method, L-arabinose transport was measured usingthe traditional ¹⁴C-labelled L-arabinose in various yeast strains withdifferent levels of arabitol formation. These experiments show that thelevel of arabitol formation corresponds well with the level ofL-arabinose uptake.

Using this method, several high arabitol producing strains have beenisolated, including two gal80 mutants and two otherwise wildtypestrains, which have 3 to 4 folds higher L-arabonise and D-galactosetransport activity than the BFY001 originally used to construct thearabinose fermenting strains. Bacterial genes encoding the araBADproteins may be introduced into these strains to achieve higher rate ofarabinose uptake and thus higher overall yield. This result alsovalidates the indirect screening method for strains with higherarabinose transport activity.

Although the present disclosure teaches the introduction of foreigngenes such as E. coil araBAD genes, into yeast cells, genes from otherspecies encoding proteins that perform the same or similar function asthe E. coli araBAD proteins, i.e., converting L-arabinose into variousintermediates and eventually into ethanol, may be used in place of theE. coli araBAD genes (See e.g., Becker and Boles, 2003, using araA fromBacillus subtilis). The DNA of the foreign genes may be present in ahost cell at one copy, or preferably, in more than one copy. The foreigngenes maybe under control of a constitutive promoter or an induciblepromoter.

The foreign genes may be present as plasmids or minichromosomes in thehost yeast cells, or alternatively, the plasmids carrying the foreigngenes may be engineered so that the foreign genes are integrated intothe chromosomes of the host through genetic recombination. In the lattercase, the foreign tends to be maintained after generations, even whenthe host cells are grown in rich media where no selective pressure ispresent. By contrast, in the former case where the genes remain on avector, the genes may be lost after a few generations. Under thosecircumstances, the host yeast cells are preferably grown in a minimummedia supplemented with appropriate amino acids or purine or pyrimidinebases so that a selective pressure helps maintain the plasmids.

Cell-free or whole-cell fermentation may be used to convert arabinose toethanol. In the whole cell fermentation process, the transformants maybe grown on minimum media with appropriate supplementation to maintainthe plasmids. The transformant cells are preferably grown on galactoseto induce the expression of galactose permease. More preferably, thecells are grown on both arabinose and galactose before the fermentationassays. In addition, transformant cells can be grown on both arabinoseand glucose before the fermentation assays (see FIG. 19).

In the cell-free fermentation system, cells are harvested and the cellsare lysed to release enzymes for the conversion of the sugar to ethanol.One bottleneck for a whole-cell fermentation system is the uptake ofarabinose by the cells, which may explain its lower overall yield ofethanol than the cell-free system. However, whole-cell fermentation ispreferred because it is easier to perform. In a whole-cell-fermentationsystem, the cells may be mixed directly with the biomass or othersubstrates, requiring no extra steps of cell-handling.

In a preferred embodiment, the various arabinose metabolic pathwaysdisclosed here may be introduced into a S. cerevisiae strain that havebeen modified to facilitate its arabinose uptake. Such strain mayinclude but are not limited to strains that express both the Gal2p andone or two of the novel arabinose transporters similar to the onesdisclosed here. The expression levels of the array of arabinosetransporters may be fine-tuned such that they are commensurate with therate of arabinose metabolism inside the engineered yeast cells. Mostpreferably, the expression levels of the transporters may be linked tothe arabinose metabolic rate in each cell, such that the arabinose istaken in more rapidly by those cells that convert arabinose to ethanolmore efficiently.

The examples herein illustrate the present instrumentalities by way ofillustration, and not by limitation. The chemicals, biological agentsand other ingredients are presented as typical components or reactants,and the procedures described herein may represent but one of the typicalways to accomplish the goal of the particular experiment. It isunderstood that various modification may be derived in view of theforegoing disclosure without departing from the spirit of the presentdisclosure.

Example 1 Cloning of the New Transporter Gene KmLAT1

A K. marxianus genomic library was constructed in our yeast -vectorpuffy13 which contains the yeast 2μ origin of replication, a URI3selection cassette, and a BamHI site located between the PGK1 promoterand GAL10 terminator. After partial digestion of 200 μg of genomic DNAwith Sau3AI restriction enzyme, fragments of 2-8 kb in length weregel-isolated and ligated into the BamHI site of pBFY013. This ligationreaction was then transformed into E. coli and plated for recovery.Plate counts produced ˜3000 cfu's/10 μl of transformed cells and theplasmid DNA from 24 colonics was screened for presence of insertrevealing 22 of 24 transformants had an insert ranging from 1 kb to ˜8kb giving an average insert size of 3.2 kb. The transformed cells werescraped from the plates, DNA recovered, and 5 μl was transformed intocompetent BFY518 cells. The strain, BFY518, was cured of the GAL2over-expression plasmid negating its ability to form colonies on agarplates containing L-arabinose as the sole carbon source enablingrestoration of colony formation by complementation with a heterologousL-arabinose transporter. To count the number of transformed yeast cells,10 μl of the yeast library transformation were plated onto minimalglucose media yet the colonies were so dense that only an estimate of˜5000 colonies was possible. The rest of the transformation mix (˜140μl)was plated onto minimal media containing 2% L-arabinose for selectionfrom which a small amount of background growth was noticed. The plateswere then replica plated to fresh L-arabinose minimal media. The totalnumber of cells plated for selection represented ˜280,000 transformantsrepresenting ˜8 fold coverage of the 10.7 mb K. marxianus genome (SeeDujon et al., 2004). Two colonies grew on the replica plates and theplasmid DNA was rescued and re-transformed into BFY518 allowing growthonce again on L-arabinose confirming that the K. marxianus genomicinsert carried on these plasmids was responsible for growth. Restrictionanalysis suggested both plasmids harbored the same insert ofapproximately 8.8 kb in size.

Example 2 Sequence Analysis of the KmLAT1Gene

Sequencing results showed that both plasmids had identical inserts of8838 kb containing two ORFs on the 5′ end of the insert. Both of theseORFs showed strong homology to yeast sugar transporters. One transporterORF was interrupted by a fragment of an unrelated ORF suggesting thatrecombination of fragments during ligation into the vector occurred inlibrary construction (FIG. 4). Recombination of library fragments duringligation into the vector was shown by PCR walking experiments performedon K. marxianus genomic DNA. Walking was performed out of thetransporter in a 5′ direction and additional transporter sequencesincluding the start codon was recovered rather than the additionalsequence from the undated ORF. The uninterrupted transporter ORF, termedKmLAT1, was recovered twice more in another subsequent libraryscreening. This ORF was 1668 bp in length and shared homology withhigh-affinity glucose transporters in particular, HGT1 from K. lactis(Table 4) and showed a much closer association with high-affinityglucose transporters from non-conventional yeasts than the bacterialaraE genes or S. cerevisiae hexose transporters (FIG. 1).

Transmembrane region prediction by the software Tmpred shows 12transmembrane regions with a larger intercellular loop between regions 6and 7 (FIG. 2) (See Hoffmann et al, 1993), typical of GAL2 and otheryeast sugar transporters having 10-12 transmembrane regions (See e.g.,Alves-Araujo et al., 2004; Day et al., 2002; Kruckeberg et al., 1996;Pina et al., 2004; and Welerstall et al. 1999).

Example 3 KmLAT1 Expressed in S. cerevisiae Enables Growth on Arabinose

The coding sequence of KmLAT1 was isolated by PCR from genomic DNA of K.marxianus and cloned into a yeast 2μ plasmid under control of the PGK1promoter of S. cerevisiae. This construct was transformed into a GAL2deleted strain of S. cerevisiae adapted to L-arabinose. Briefly, cellsare grown in appropriate selective glucose minimal media untilsaturation then washed and diluted to a starting OD₆₀₀ of 0.2 in minimalmedia supplemented with 2% L-arabinose. Cultures are incubated untilexponential growth Is observed then the cultures are diluted twice intothe same media for continued growth to establish the final L-arabinoseutilizing adapted strain which is purified on streak plates. Controlplasmids carrying the yeast GAL2 gene and an empty vector were also usedto transform yeast cells.

Yeast cells with a 2μ plasmid carrying the KmLAT1 or GAL2 gene or cellswith an empty 2μ plasmid were grown with shaking in liquid minimum mediacontaining 2% L-arabinose as the sole carbon source. The OD₆₀₀ of eachculture was measured and monitored by 140 hours. Growth curve resultsshow that KmLAT1 is sufficient to support growth on L-arabinose whencompared to cells harboring the empty vector which does not show anysigns of growth (FIG. 5). This result confirms that the KmLAT1 geneencodes an arabinose transporter that enables yeast cells to grow onL-arabinose.

Example 4 Comparison of the Arabinose Transport Kinetics between Gal2and KmLAT1 Expressed in S. cerevisiae

The transport characteristics of the KmLAT1 and tire Gal2 transportersexpressed in S. cerevisiae were compared. Both transporters wereexpressed in a host background adapted for growth on L-arabinose inwhich the endogenous copy of GAL2 had been entirely replaced with a HIS3selection marker. The KmLat1 transporter showed a low-affinitytransporter having a K_(m)=230 mM and a V_(max)=55 nmol/mg·min (FIG.6A). This is in contrast to the high-affinity active transport activityinduced in the wild type K. marxianus when grown on 2% L-arabinose (FIG.6B). These results suggest there are at least 2 transporters in K.marxianus that may transport L-arabinose but just the high-affinityactivity is induced in the wild type when grown 2% L-arabinose.Inhibition experiments showed that when KmLAT1 is expressed in S.cerevisiae it is not significantly inhibited by protonophores such asNaN₃, DNP, and CCP. Neither is KmLAT1 inhibited by H+-adenosinetriphosphatase (ATPase) inhibitors such as DESB and DCCD (Table 6). Thisis in contrast to the transport activity in wild type K. marxianus,suggesting that KmLAT1 is a facilitated diffusion permease similar tothe Gal2 permease. Competition experiments showed that KmLAT1 isout-competed by glucose, galactose, xylose, and maltose when expressedin S. cerevisiae (Table 6).

TABLE 6 Effect of Inhibitors or Competing Sugars on the Rate ofL-Arabinose Transport in L-Arabinose-Grown S. cerevisiae Expressing GAL2or KmLAT1 Relative L-arabinose Inhibitor or Concentration transport (%)Competing Sugar (mM) Gal2 KmLat1 None NA 100^(a ) 100^(c ) NaN₃ 10 66 11CCCP 5 46 61 DCCD 5 69 55 DNP 5 72 75 DESB 5 81 100  None NA 100^(b )100^(d ) Glucose 900 10 17 Galactose 900  3 23 Xylose 900 25 25 Maltose450 ND 38 ^(a)Uptake rate was 66.0 nmol mg⁻¹ min⁻¹ determined with 118mM labeled L-arabinose. ^(b)Uptake rate was 18.9 nmol mg⁻¹ min⁻¹determined with 30 mM labeled L-arabinose. ^(c)Uptake rate was 7.7 nmolmg⁻¹ min⁻¹ determined with 118 mM labeled L-arabinose. ^(d)Uptake ratewas 3.6 nmol mg⁻¹ min⁻¹ determined with 30 mM labeled L-arabinose. ND,Not Done.

Transport kinetics of S. cerevisiae BFY597 over-expressing the Gal2permease grown on 2% L-arabinose showed a of K_(m) of 550 and a V_(max)of 425 nmol/mg·min for L-arabinose transport (FIG. 6A). Inhibitionassays showed a reduction but not a complete inhibition of transportsuggestive of facilitated diffusion transport (Table 6). Competitionstudies showed that glucose, galactose, and xylose significantly reducedL-arabinose transport indicating that those sugars are preferentiallytransported over L-arabinose (Table 6). The kinetics of galactosetransport were also measured in this strain and indicate that Gal2p hasa K_(m) of 25 mM and a V_(max) of 76 nmol/mg·m for galactose transport(data not shown) demonstrating a higher affinity for galactose thatwould out-compete L-arabinose for transport.

Example 5 Cloning of the New Transporter Gene PgLAT2

Wildtype Pichia guilliermondii NRRL Y-2075 was obtained from theAgricultural Research Service Culture Collection (NRRL). Pichiaguilliermondii cells were grown in minimal media supplemented with 2%L-arabinose, galactose, or xylose. Cells were collected in mid-growthand washed twice in water before suspension in water at about 30 mg/ml.RNA was extracted from the cells using the acid phenol method (Ausubel,et al., Short Protocols in Molecular Biology, John Wiley and Sons,1999). Briefly, approximately 15 mL of fresh culture was added to about25 mL of crushed ice and centrifuged at 4° C. for 5 min at 3840×g. Cellswere washed twice with cold DEPC-treated water, and the pellets werefrozen at −80° C. After the pellets were resuspended in 400 ul TES (10mM Tris HCl, pH 7.5, 5 mM EDTA, 0.5% SDS), 400 ul of acid phenol wasadded. The samples were vortexed vigorously for 10 sec, followed byincubation for 30-60 min at 65° C. with occasional vortexing. The tubeswith the samples were then chilled on ice and spun for 5 min at 4° C.The aqueous phase was removed and re-extracted with chloroform. Theaqueous phase was then ethanol precipitated using 0.1 volume of 3 Msodium acetate (pH 5.3) and two volumes of 100% ethanol. The pellet waswashed using 80% ethanol, dried, and resuspended in 50 ul DEPC H₂O.Total RNA concentration-was quantitated by measuring the OD₂₀₀ andvisualized on agarose gels.

RNA purification, synthesis of cDNA, and differential display wereperformed at GenHunter Corporation according to standard techniques. DNABands showing higher levels of expression from arabinose-grown cellsrelative to xylose- or galactose-grown cells were reamplified using thedifferential display amplification primers. Direct sequencing wasperformed on the PCR products using the GenHunter arbitrary primers. Incases that did not yield clean sequence, the amplification products werecloned in the TOPO-TA vector pCR2.1 (Invitrogen) and individual cloneswere sequenced. Sequences were then compared to the databases usingBLASTX analysis and those that showed similarity to known transporterson transporter-like proteins were examined further. One of thesesequences led to the identification of a novel transporter gene, PgLAT2from Pichia guilliermondii, PgLAT2 gene has an ORF of 1617 nucleotidesencoding a protein with a predicted length of 539 amino acids (FIG. 7).

Example 6 Characteristics of Sugar Transport by Pichia guilliermondii

The induction of L-arabinose transport in wild type P. guilllermondiiwas examined. Wildtype Pichia guilliermondii cells were grown in minimalmedia supplemented with 2% L-arabinose, galactose, or xylose whileBFY605 cells were grown in the same media supplemented with 0.2%L-arabinose. Cells were collected in mid-growth and washed twice inwater before suspension in water at about 30 mg/ml. Uptake ofL-(1-¹⁴C)arabinose (54 mCi/mmol, Moravek Biochemicals Inc.),D-(1-¹⁴C)galactose (57 mCi/mmol, Amersham Biosciences), orD-(1-¹⁴C)xylose (53 mCi/mmol, Moravek Biochemicals Inc.) was measured aspreviously described (Stambuk, Franden et al. 2003). Assays wereperformed in 5, 10, or 30 second periods to maintain initial rates.Appropriate experiments ensured uptake was linear for at least 1 minute.Transport activity was described as nmoles of labeled sugar transportedper mg cell dry weight per minute. Inhibition and competition assayswere performed as previously described (Stambuk, Franden et al. 2003).

Cells grown on L-arabinose were able to transport L-arabinose whereascells grown on galactose or xylose were not able to transportL-arabinose. Additionally, xylose transport was about double in cellsgrown in L-arabinose media compared to cells grown in xylose media.Galactose was transported at the same rate independent of growthsubstrate (FIG. 8). Transport competition between L-arabinose and xylosewas also examined. Uptake of labeled L-arabinose was reduced by 96% when100 × un-labeled xylose was included in the transport assay whereasuptake of labeled xylose was only reduced by 16% when 100 × un-labeledL-arabinose was included in the assay (FIG. 9). This data suggests thatin P. guilliermondii, growth on L-arabinose induces expression of aspecific transport system capable of transporting L-arabinose andxylose. Additionally, this system preferentially transports xylose atthe expense of L-arabinose if both sugars are present and has a highertransport velocity for xylose than the transport system induced whengrown on xylose. By contrast, transport activity for L-arabinose is notinduced when grown on xylose.

Example 7 Arabinose Transport Kinetics of PgLAT2 Expressed in S.cerevisiae

The L-arabinose transport characteristics of the PgLAT2 transporterexpressed in S. cerevisiae grown on 0.2% L-arabinose medium showed thesame L-arabinose transport characteristics as wildtype P. guilliermondii(FIG. 10). The PgLAT2 transporter when expressed in S. cerevisiae has aK_(m)=0.07 mM and V_(max)=18 nmol/mg·min. Inhibition experiments showedsignificant inhibition of transport by protonophores (NaN₃, DNP, andCCP) and H+-adenosine triphosphatase (ATPase) inhibitors (DESB and DCCD)similar to the inhibition Observed in wildtype P. guilliermondii (Table7). Compaction experiments showed that L-arabinose uptake by the PgLAT2transporter was inhibited by glucose, galactose, xylose and to a lesserextent by maltose (Table 7).

TABLE 7 Effect of Inhibitors or Competing Sugars on the Rate ofL-Arabinose Transport in L-Arabinose-Grown P. guilliermondii Y-2075 andS. cerevisiae BFY605 Inhibitor or Relative L-arabinose transportCompeting Concentration S. cerevisiae (PgLAT2 Sugar (mM) P.guilliermondii transporter) None^(a) — 100 100 NaN₃ 10 1 16 DNP 5 0 4CCCP 5 0 2 DCCD 5 22 36 DESB 5 8 1 None^(b) — 100 100 Glucose 120 ND 17Galactose 120 ND 20 Xylose 120 4 0 Maltose 120 ND 30 ^(a)Rate ofL-arabinose transport was 11.2 nmol mg⁻¹ min⁻¹ for P. guilliermondii and10.4 nmol mg⁻¹ min⁻¹ for S. cerevisiae (PgLAT2 transporter) determinedwith 0.33 mM labeled L-arabinose. ^(b)Rate of L-arabinose transport was14.2 nmol mg⁻¹ min⁻¹ for P. guilliermondii and 14.4 nmol mg⁻¹ min⁻¹ forS. cerevisiae (PgLAT2 transporter) determined with 1.2 mM labeledL-arabinose.

The transport activities, inhibition profiles, and competition rateswith respect to xylose of wildtype P. guilliermondii and of the PgLAT2transporter expressed in S. cerevisiae are identical suggesting that P.guilliermondii has a single, high affinity, active transporter chargedwith uptake of L-arabinose. There are no L-arabinose transportactivities that are unaccounted which suggests the presence of a singleL-arabinose transporter in P. guilliermondii.

Example 8 Synergistic Effect on Growth Rate and Sugar Utilization by S.cerevisiae Expressing Gal2p and the New Transporter Proteins-PgLat2 andKmLat1

To determine the complementary effects on arabinose transport by thethree transporters, namely, Gal2p, PgLat2 and KmLAT1, yeast strains wereconstructed with appropriate selection markers to allow differentpathway and transporter combinations to be expressed. All possibletransporter combinations were generated by introducing transporterexpression plasmids for PgLat2 and KmLat1 (or empty vectors) into S.cerevisiae strains expressing the bacterial genes araA; araB and araD(See e.g., Becker and Boles). All strains expressing Gal2p (due to thegal80A genotype), plus or minus other transporters, were able to grow on2% or 0.2% L-arabinose after extensive lag times (a process termed“adaptation.”). A relatively low concentration of L-arabinose (0.2%) wasused in this experiment as strain differences are more pronounced atthis concentration. Once “adapted” to growth on 0.2% L-arabinose, thestrains were able to grow more quickly and growth curves for eachtransporter combination were generated.

FIG. 11 shows a comparison of shake flask-growth curves for four strainson 0.2% L-arabinose, all of which express Gal2p (via the GAL80 deletion)in the absence or presence of the novel transporter PgLat2 (also seeTable 5). A significant lag time was observed due to their inoculationfrom stationary cultures. However, once growth initiated, the growthrate was relatively rapid. The doubling time for each culture in theexponential phase of the curve is shown in Table 5. The doubling timefor the PgLat2 and Gal2p co-expressing cells was markedly shorter thanin the cells expressing only Gal2p (15 hours vs. 19 hours). A secondobservation relates to the overall extent of growth. The PgLat2expressing strain appeared to grow to a higher overall optical densityat saturation, suggesting that this strain was able to utilize thecarbon source (L-arabinose) in the medium more completely (FIG. 11).

Example 9 Co-expression of Gal2p with PgLAT2 or KmLAT1 Enables moreComplete Utilization of Arabinose by Recombinant S. cerevisiae

Doubling limes for the cultures described above in Example 8 weremeasured in early exponential phase for each culture. Doubling time wasmeasured by the period of time taken for the number of cells to doublein a given cell culture (See generally, Guthirie and Fink, 1991). Theconcentration of remaining L-arabinose at the 276 hour time point wasdetermined by HPLC (for saturated cultures only). The concentration ofL-arabinose in the starting media was about 1.89 g/L and theconcentration of L-arabinose in media without L-arabinose had anundetectable level (<0.1 g/L). As shown in Table 5, significantly lessresidual L-arabinose remained in the culture of cells expressing bothGal2p and PgLAT2 than in the culture of cells expressing Gal2p alone.

Example 10 Construction of S. cerevisiae Strain Deficient in AldoseReductase (AR)

Based on the sequence of a presumptive AR gene, two oligonucleotideprimers were designed and the AR gene along with 600 bp of flanking DNAwere cloned by PCR using genomic DNA isolated from yeast as template.Using another set of primers, an AR deletion construct was made in whichall the coding sequences of the AR gene were replaced with a restrictionenzyme site (SalI). The yeast LEU2 gene was isolated as a SalI-XhoIfragment and cloned into the SalI site of the AR deletion construct. TheDNA fragment containing the LEU2 gene and AR flanking sequences was usedto transform the leu2⁻ yeast strain BFY001. LEU⁺ transformants wereisolated, grown and analyzed by Southern and PCR analysis to confirmthat the AR gene in the genome had been deleted and replaced with theLEU2 gene by homologous recombination.

One such transformant, designated BFY002, was chosen as a host forfurther construction of arabinose fermenting yeast strains. Shake-flaskexperiments were conducted and the results showed that arabitolformation in BFY002 had been reduced to about 50% in comparison with theparental strain BFY001.

Growth of BFY002 on glucose and xylulose was compared with that ofBFY001. Briefly, yeast strains BFY001 and BFY002 were grown in richmedium YPD. Cells were collected by centrifugation and washed withsterile water. The washed cells were suspended in water at the originaldensity. 50 μl of this cell suspension was used to inoculate 5 ml ofmedium containing yeast nitrogen base (“YNB”) supplemented with leucine(“Leu”), tryptophan (“Trp”), histidine (“His”) and uracil (“Ura”), pluswith 1% glucose or 1% xylulose. The growth of each strain on both mediaat 30° C. with shaking was monitored by measuring the OD₆₀₀ for about 6generation times. As expected, both strains had much shorter doublingtime on glucose than on xylulose. No significant difference in thegrowth curves was observed between BFY001 and BFY002 regardless ofwhether glucose or xylulose was used (FIG. 12A and FIG. 12B).

Example 11 Isolation of E. coli araBAD Genes and B. subtillis araA andIntroduction into S. cerevisiae

Primers were designed to isolate E. coli araA gene as a BgIII fragment,and the araB and araD genes were isolated as BamHI fragments by PCR fromplasmid pZB206. The fragments containing the three genes were clonedinto a yeast expression vector pBFY004, which contains PGK promoter,GAL10 terminator, and TRP1 selection marker. Each plasmid carryingindividual gene was then transformed into the yeast strain BFY002 inseparate experiments. Transformants carrying individual plasmid wereanalyzed for the expression levels of each ara protein. L-ribulokinase(araB) and L-ribulose-5-P-4-epimerase (araD) were expressed at a higherlevel while L-arabinose isomerase (araA) was expressed at a lower level.

In order to introduce all three ara genes into the same cell, URA3 andHIS3 expression vectors for each ara gene were constructed byre-engineering the TRP1 plasmid, pBFY004. Briefly, the TRP1 codingsequence was removed and replaced with a SalI restriction site togenerate a plasmid designated as pBFY011. Other selection markers, HIS3or URA3, were then cloned into this plasmid. Another strategy forintroducing all three ara genes into the same cell was to construct aplasmid carrying all three genes. Briefly this was done by combiningeach ara gene with a different promoter/terminator combination toprevent homologus recombination and thus loop-out of the genes withcorresponding loss of function. Primers were designed to clone the E.coli araD gene between the TDH3 promoter and GAL2 terminator. Thisexpression cassette was then moved to pBFY007 (which already has the E.coli araA gene cloned between the PGK1 promoter and GAL10 terminator)and designated pBFY051. Similarly primers were designed to clone the E.coli araB gene between the PGII promoter and the PDC1 terminator. Thisexpression cassette was then moved to pBFY051 to create pBFY090 whichnow has all three E. coli ara genes. The B. subtilis araA gene was thenisolated using PCR and cloned between the PGK1 promoter and GAL10terminator to replace the E. coli araA. The URA3 gene was isolated as aSalI fragment and cloned into the SalI site to construct the plasmidpBFY012. Similarly, a HIS3 expression vector, pBFY013, was contructed byengineering and cloning the HIS3 gene into pBFY011.

The engineered ara genes were cloned into each of these expressionvectors to generate a series of expression vectors carrying each of thearaBAD genes with either Trp1, Ura3 or His3 markers (Table 3; also seeFIG. 13A, FIG. 13B, FIG. 13C, FIG. 14A, FIG. 14B and FIG. 14C).Appropriate combinations of these expression vectors were introducedinto the strain BFY002. Similarly, the plasmid containing all three aragenes was introduced into Hip strain BFY057. The transformants werecharacterized and assayed for growth and fermentation of arabinose.

Example 12 Determination of the Copy Numbers of Plasmids Carrying araBADin S. cerevisiae

The copy numbers of the three plasmids present m the strain BFY013(Table 8) transformed as described in Example 11 were determined. Oncolony of BFY013 was isolated and used to inoculate a flask with yeastminimum medium. The cells were allowed to grown to exponential phase andthe cells were harvested by centrifugation. Spheroplasts of the cellwere prepared and DNA was extracted from these spheroplasts (See Guthrieand Fink, 1991). E. coli strain DH5α cells were then transformed withthe extracted yeast DNA. Bacterial transformants were plated out andplasmid DNA in individual colonies was isolated and characterized byrestriction digest followed by agarose, gel electrophoresis. Assumingthat the individual plasmids carrying each of the araBAD genes possessedthe same capability to transform bacterial cells, the ratio between thecopy numbers of each plasmids present in the original yeast cells wasestimated based on the number of E. coli transformants harboring eachplasmid (Table 8).

TABLE 8 Ratio of the 3 plasmids in BFY013 ara gene Number araB 11 araA 4araD 15

Example 13 Assays of Enzymntic Activities of araBAD Proteins Expressedin S. cerevisiae

The activities of the three E. coli enzymes heterologously expressed inS. cerevisiae were measured in the crude extracts of the yeasttransformants according to protocol described in Becker and Boles, 2003.The results of these assays are summarized in Table 9. Table 10 comparesthe enzymatic activities of the two strains used for subsequentfermentation. The enzymatic assays were performed in the presence ofabsence of 20 mM MnCl₂, but it appears that MnCl₂ does not havesignificant effect on the overall results (Table 10).

TABLE 9 Enzyme activities in transformants carrying all 3 ara genesL-arabinose isomerase (araA) L-ribulokinase (araB) L-ribulose5-P4-epimerase (araD) Sp. act umol/min/mg Sp. act umol/min/mg Sp. actumol/min/mg Strain Oct 20-21 (1) Oct 20-21 (2) Dec 20-22 (2) Oct 20-21Dec 20-22 Oct 20-21 Dec 20-22 BFY012 nd nd nd nd nd nd BFY013 0.05 0.100.11 trp 1.2 1.3 ura 0.5   1.0 his BFY014 0.04 0.11 0.11 trp 1.2 1.9 hisnd nd ura BFY015 nd nd ura 2.4 2.8 trp 0.39  0.9 his BFY016 0.03 0.06his 2.4 2.4 trp nd nd ura BFY017 0.02 nd his 0.9 1.1 ura 1.56 >3.5 trpBFY018 0.01 nd ura 1.2 1.0 his 1.12 >2.2 trp Zymomonas 0.85 0.9 1.9  nd= not detected (1) = cysteine-carbozole (2) = new method, NADHdisappearance All cultures were grown in selective medium (YNB + 2%glo-trp-his-ura)

TABLE 10 Enzyme Activities in Strains Used for Fermentation IsomeraseKinase Epimerase (araA) (araB) (araD) Sp. act Sp. act Sp. act MnCl2Protein umol/ umol/ umol/ Strain (20 mM) mg/ml min/mg min/mg min/mgBFY012 No 6.8 nd nd nd BFY012 Yes 6.1 nd nd nd BFY013 No 7.0 0.09 2.20.6 BFY013 Yes 6.7 0.09 1.9 0.5

Example 14 Whole-cell Fermentation in S. cerevisiae

The transformed yeast cells carrying bacterial genes araBAD were firstgrown in galactose or arabinose alone or in the presence of bothgalactose and arabinose in YNB. Cells were collected by centrifugationand washed in water. The washed cells were resuspended in liquidmedia-containing 1% yeast extract and 2% peptone (YP). The cellsuspensions were aliquoted into various tubes before appropriate sugarswere added. The tubes were incubated at 30° C. and cell samples weretaken at the time indicated. The samples were filtered and analyzed forethanol concentration by gas chromatography (GC) according to Tietz,1976 (Table 11 and FIG. 15) or by high performance liquid chromatography(HPLC). As shown in Table 11, cells that were grown in both galactoseand arabinose immediately before the fermentation assay had a slightlyhigher overall yield of ethanol than cells grown in galactose aloneduring the same period. The strains BFY534 and BFY535 were grown inarabinose alone prior to fermentation. From a starting concentration of19 g/L of L-arabinose, BFY534 and BFY535 used 12.7 and 11.8 g/L ofL-arabinose to yield 4.7 and 4.9 g/L of ethanol in 48 hoursrespectively. The percentage of maximum theoretical conversion wouldthus be 75% and 78% respectively and a productivity of 0.012 g EtOH/gcells hr for both strains. In an additional fermentation with strainBFY534 performed in shake flasks, 19.7 g/L of L-arabinose was convertedto 8.5 g/L ethanol in 96 hrs giving 85% of the theoretical maximumconversion and a productivity of 0.017 g EtOH/g cells hr.

TABLE 11 Ethanol Concentration (g/l) from Whole Cell FermentationGlucose Arabinose (66.7 g/l) (66.7 g/l) No sugar Time (hrs) 0 24 0 24120 0 24 120 BFY012 (Gal) 1.9 33.6 1.0 1.1 1.8 1.0 0.9 1.8 BFY013 (Gal)1.6 33.3 0.9 1.3 2.6 0.0 0.9 0 BFY015 (Gal) 1.5 33.8 0.9 1.3 2.5 0.0 0.91.3 BFY012 (G + A) 2.1 34.3 1.0 1.2 1.9 1.0 1.0 1 BFY013 (G + A) 1.734.2 1.0 1.8 4.2 0.9 1.0 0 BFY015 (G + A) 1.5 33.8 0.9 1.5 3.2 0.0 1.0 0

Example 15 Cell-free Fermentation in S. cerevisiae

For fermentation of arabinose in a cell-free system, yeast transformantswere grown in the presence of both galactose and arabinose in YNB. Cellswere collected by centrifugation and washed in water. Cell walls wereremoved by enzymatic digestion and the cells were then lysed in a lysisbuffer containing 20 mM potassium phosphate buffer, pH 7 and 10 mM MgCl₂and 1 mM DTT. Cells debris were removed by centrifugation, and thesupernatant was transferred to a tube where various chemicals were addedto the supernatant such that the fermentation mix contained 7 mM Mgacetate, 5 mM ATP, 0.1 mM diphosphoglyceric acid, 4 mM Na arsenate and 2mM NAD⁺. Fermentation was started by adding appropriate sugar to thefermentation mix in the tube. The tube was incubated at 30° C. andsamples were taken at the time indicated. The samples were boiled,centrifuged, filtered and analyzed for ethanol concentration by gaschromatography (GC) as previously described. Results of the cell-freefermentation are shown in Table 12, FIG. 16A, FIG. 16B, FIG. 16C, FIG.17A, FIG. 17B, FIG. 17C and FIG. 18.

TABLE 12 Ethanol Concentration (g/l) from Cell-Free Fermentation GlucoseArabinose No sugar Time (hrs) 0 2 24 48 72 0 2 24 48 72 0 2 24 48 72BFY012 0.0 2.7 8.1 8.3 8.2 0.0 1.4 1.9 1.9 1.9 1.2 1.5 1.9 1.9 2.0BFY013 0.0 3.1 12.6 13.2 12.8 0.0 1.7 4.5 6.0 6.8 0.0 1.5 2.1 2.1 2.0BFY012 (20 mM MnCl₂) 1.3 2.6 9.2 9.0 8.5 1.2 1.4 1.9 1.9 2.1 0.0 1.4 2.02.0 1.8 BFY013 (20 mM MnCl₂) 1.3 3.0 12.8 13.1 — 1.3 1.8 4.6 6.1 6.8 1.31.6 2.0 2.0 —

Example 16 Mixed-Sugar Fermentation in S. cerevisiae

Yeast attains adapted to contain the araB and araD genes from E. coli,the araA isomerase gene from B. subtilis, and the GAL2 overexpressionplasmid have been described previously (see for example, Example 11);Adapted strains, BFY534 for example, were tested for fermentation ofglucose, arabinose, and a mixture of arabinose and glucose. Inparticular, 125 ml non-baffled flasks containing 50 ml of yeast-extractpeptone media (including adapted yeast) and either no sugar, glucose,L-arabinose, or both glucose and L-arabinose were prepared. The flaskswere closed with Saranwrap held in place with rubber bands. Thefermentations were performed a 30° C. with gentle shaking (80 rpm). Inall cases, each sugar was present at a concentration of 20 g/L.

The results are illustrated in FIG. 19, where a greater than 50%increase in ethanol production was obtained in co-fermentation ofglucose and L-arabinose, compared to the ethanol yield of glucose alone.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permulations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permulations, additions and sub-combinations as are within their truespirit and scope.

This specification contains numerous citations to references such aspatents, patent applications, and scientific publications. Each ishereby incorporated by reference for all purposes.

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1-37. (canceled)
 38. A transgenic yeast strain for fermenting arabinoseto ethanol, the transgenic yeast strain comprising genes for theexpression of an araA peptide, an araB peptide and an araD peptide andwherein the transgenic strain is aldose reductase deficient (AR⁻). 39.The transgenic yeast strain of claim 38, wherein the araA gene isderived from E. coli or B. subtilis.
 40. The transgenic yeast strain ofclaim 38, wherein the transgenic yeast strain has a deletion ordisruption of the aldose reductase gene.
 41. The transgenic yeast strainof claim 40, wherein the transgenic yeast strain has a deletion ordisruption of the open reading frame (ORF) yhr104w.
 42. The transgenicyeast strain of claim 38, wherein the transgenic yeast strain is capableof producing at least 4.7 g/liter of ethanol from 19 g/L of arabinose.43. The transgenic yeast strain of claim 38, wherein the transgenicyeast strain has a mutation of the aldose reductase gene.
 44. Thetransgenic yeast strain of claim 38, wherein the yeast strain includesat least one arabinose transporter gene selected from the groupconsisting of GAL2, KmLAT1 and PgLAT2.
 45. An ethanol producing yeaststrain, comprising at least one heterologous arabinose transporterwherein the ethanol producing yeast strain is aldose reductase (AR)deficient (AR-).
 46. The ethanol producing yeast strain of claim 45,further comprising at least two heterologous arabinose transporters. 47.The ethanol producing yeast strain of claim 45, wherein the heterologousarabinose transporter is from a non-conventional yeast species.
 48. Theethanol producing yeast strain of claim 47, wherein the heterologousarabinose transporter is KmLat1 or PgLat2.
 49. The ethanol producingyeast strain of claim 45, wherein the yeast strain overexpresses aGAL2-encoded galactose permease.
 50. The ethanol producing yeast strainof claim 49, wherein the yeast derived GAL2-encoded galactose permeaseis under control of a TDH3 promoter.
 51. The ethanol producing yeaststrain of claim 45, further comprising one or more bacterial genes thatfacilitate arabinose utilization and fermentation in the ethanolproducing yeast strain.
 52. The ethanol producing yeast strain of claim51, wherein the one or more bacterial genes for facilitated arabinoseutilization and fermentation are selected from the group consisting ofaraA, araB and araD.
 53. The ethanol producing yeast strain of claim 51,comprising araA, araB and araD.
 54. The ethanol producing yeast strainof claim 52, wherein at least one of araA, araB and araD is derived fromE. coli.
 55. A method for producing ethanol utilizing a transgenic yeaststrain, comprising the steps of: providing a material comprisingarabinose, performing a fermentation of the material using thetransgenic yeast strain, wherein the transgenic yeast strain comprisesgenes for the expression of an araA peptide, an araB peptide and an araDpeptide and wherein the transgenic yeast strain is aldose reductasedeficient (AR⁻); and producing at least 8 g/liter ethanol.
 56. Themethod of claim 55, wherein the material comprises arabinose andglucose, and the method produces at least 17 g/liter ethanol.
 57. Themethod of claim 55, wherein the transgenic yeast strain has a deletionor disruption of the aldose reductase gene.